T cell responses against dominant tumor antigens (TA) can kill tumor cells but are less effective against poorly immunogenic (subdominant) TAs. Therefore, strategies are needed to induce potent and long-lasting T cell responses against dominant and subdominant TAs. Toll-like receptor (TLR) agonists help to generate potent antitumor T cell responses by stimulating TLRs on antigen presenting cells (APCs). Stimulation of these receptors induces the production of inflammatory cytokines and increases the expression levels of costimulatory ligands necessary for optimal T cell activation. Whereas the roles for TLRs on APCs are clear surprisingly, little is known about the effects of TLR engagement on CD8 T cells. Our group has generated new data demonstrating that TLR2 engagement on TA-specific CD8 T cells increases T cell proliferation. TLR2 stimulation also increases IFN-?, granzyme B, and perforin production and boosts cytolytic activity following activation with poorly immunogenic TAs in vitro. Treatment of tumor-bearing mice (wild type or TLR2 knock out) with adoptive T cell transfer (ACT) of tumor-reactive CD8 T cells and TLR2 ligand induces the regression of established tumors and generates de novo T cell responses to various TAs. In contrast, treatment with TLR2 ligand and TLR2 knock out (-/-) CD8 T cells does promote significant tumor regression. Our hypothesis is that activating TLR2 signals in TA-specific CD8 T cells leads to potent and long-lived antitumor activity by potentiating responses to weakly immunogenic TAs. The first objective of this proposal is to achieve a mechanistic understanding of how activating TLR2 signals in CD8 T cells enhances activation to poorly immunogenic TAs. The second objective is to determine the in vivo cellular and antitumor responses of TLR2-ligated CD8 T cells. The specific aims are: (1) Define the signaling pathway through which TLR2 signals in CD8 T cells potentiate activation to poorly immunogenic tumor antigens. (2) Determine the responses of TLR2-stimulated CD8 T cells following activation with a poorly immunogenic tumor antigen in vivo, including the expansion/contraction kinetics, kinetic expression of activation markers, and CTL effector function. (3) Determine the effect of TLR2- stimulated CD8 T cells on the development of effective antitumor responses and the induction of new tumor-specific T cells. We will use a physiologically-relevant mouse model to evaluate the responses of TLR2- stimulated TA-specific CD8 T cells that recognize the mouse TA gp100, referred to as pmel T cells. We will also examine the antitumor responses of TLR2-/- pmel and pmel T cells lacking the TLR2 signaling adopter molecule MyD88. In addition, we will determine the antitumor effects of TA-specific T cells engineered to overexpress TLR2 or MyD88. We envision these studies will make possible new approaches for the development of effective T cell-based therapies against cancer through a greater understanding of molecular signals that enhance T cell activation to weakly immunogenic TAs.